Synthesis of the l-Alanyl-l-alanine Cross-bridge of Enterococcus faecalis Peptidoglycan

Abstract

The enzymatic synthesis of the complete L-alanyl 1 -Lalanine 2 side chain of the peptidoglycan precursors of Enterococcus faecalis was obtained in vitro using purified enzymes. The pathway involved alanyl-tRNA synthetase and two ligases, BppA1 and BppA2, that specifically transfer alanine from Ala-tRNA to the first and second positions of the side chain, respectively. The structure of the UDP-N-acetylmuramoyl-L-Ala-␥-D-Glu-LLys(N ⑀ -L-Ala 1 -L-Ala 2 )-D-Ala-D-Ala product of BppA1 and BppA2 was confirmed by mass spectrometry (MS) and MS/MS analyses. The peptidoglycan structure of the wild-type E. faecalis strain JH2-2 was determined by tandem reverse-phase high-pressure liquid chromatography-MS revealing that most muropeptides contained two L-alanyl residues in the cross-bridges and in the free N-terminal ends. Deletion of the bppA2 gene was associated with production of muropeptides containing a single alanyl residue at these positions. The relative abundance of monomers, dimers, trimers, and tetramers in the peptidoglycan of the bppA2 mutant indicated that precursors containing an incomplete side chain were efficiently used by the DD-transpeptidases in the crosslinking reaction. However, the bppA2 deletion impaired expression of intrinsic ␤-lactam resistance suggesting that the low affinity penicillin-binding protein 5 did not function optimally with precursors substituted by a single alanine.Variations in the structure of peptidoglycan from Grampositive bacteria involve mainly the third and fifth (C-terminal) positions of the pentapeptide stem linked to the disaccharide GlcNAc-MurNAc 1 (see Fig. 1 (Fig. 1). Factors essential for methicillin resistance (fem) in S. aureus include the femA and femB genes that are required for synthesis of the pentaglycine side chain of the peptidoglycan precursors (4).In the late 1960s, the ligases for addition of glycine and L-amino acids to the ⑀-amino group of L-lysine were shown to use aminoacyl-tRNAs as substrates, whereas D-amino acids are added in a tRNA-independent reaction (5, 6). More recently, the ligase for incorporation of the first L-alanine of the side chain of the E. faecalis (7) and Weissella viridescens (8) peptidoglycan precursors has been identified based on cloning of fem-related genes in Escherichia coli and assays of the purified gene products for UDP-MurNAc-pentapeptide:L-alanine ligase activity. The assay developed in our laboratory relies on in vitro synthesis of Ala-tRNA by the purified E. faecalis alanyl-tRNA synthetase and subsequent transfer of L-Ala from the aminoacyl-tRNA to the nucleotide precursor UDP-MurNAc-L-Ala-␥-D-Glu-L-Lys-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) (7). In the current study, we used this approach to identify the ligase for incorporation of the second L-alanine in the side chain of E. faecalis peptidoglycan precursors. We report in vitro synthesis of the complete L-alanyl-L-alanine side chain, deletion of the gene encoding the ligase for incorporation of the second L-alanine from the chromosome of E. faecalis JH2-2, and the analys...

Application of a bacterial two-hybrid system for the analysis of protein–protein interactions between FemABX family proteins

“…zooepidemicus. The protein-protein interactions we were able to show add to the more-detailed knowledge about the FemABX protein family that has been gained in recent years (Benson et al, 2002;Bouhss et al, 2002;reviewed by Rohrer & BergerBächi, 2003). Proteins of the FemABX family are a recognized target for the development of novel antimicrobial agents (Kopp et al, 1996).…”

Synthesis of Mosaic Peptidoglycan Cross-bridges by Hybrid Peptidoglycan Assembly Pathways in Gram-positive Bacteria

“…Purified fractions (3 l) of reduced disaccharide peptides or lactoyl peptides were directly injected into the mass spectrometer using HPLC pumps at a flow rate of 0.2 ml/min (acetonitrile 50%, water 49.5%, formic acid 0.5%, per volume). The data were acquired with a capillary voltage of 5,200 V and a declustering potential of 20 V. The mass scan range was from m/z 400 to 2,500, and the scan cycle was 1 s. Structure assignment of muropeptides based on mass was performed as previously described (14).…”

“…Derivatives of expression vector pNJ2 harboring fem and pbp genes (see below) were introduced into E. faecalis JH2Sm::Tn916 (17) by electroporation and transferred by conjugation to E. faecalis JH2-2 (18), E. faecalis JH2-2⌬bppA2 (14), E. faecalis JH2-2⌬pbp5 (17), E. faecium D344S (19), and E. faecium BM4107 (20), as previously described (17). Spectinomycin (60 g/ml) was used in all experiments to counter select loss of the plasmids.…”

“…Inactivation of bppA1 encoding the transferase for incorporation of the first residue of the L-Ala-LAla side chain in E. faecalis has not been obtained (14). Deletion of bppA2 led to production of precursors substituted by a single L-Ala and to impaired expression of intrinsic ␤-lactam resistance (14). For these reasons, transferases of the Fem family are considered to be potential targets for the development of novel antibiotics active against ␤-lactam-resistant Gram-positive cocci (15).…”

“…Similarly, the side chain is essential for penicillin resistance in Streptococcus pneumoniae (13). Inactivation of bppA1 encoding the transferase for incorporation of the first residue of the L-Ala-LAla side chain in E. faecalis has not been obtained (14). Deletion of bppA2 led to production of precursors substituted by a single L-Ala and to impaired expression of intrinsic ␤-lactam resistance (14).…”